U.S. patent number 11,155,781 [Application Number 15/351,124] was granted by the patent office on 2021-10-26 for maintenance of differentiated cells with laminins.
This patent grant is currently assigned to BIOLAMINA AB. The grantee listed for this patent is BioLamina AB. Invention is credited to Anna Domogatskaya, Karl Tryggvason, Karl Kristian Tryggvason.
United States Patent |
11,155,781 |
Tryggvason , et al. |
October 26, 2021 |
Maintenance of differentiated cells with laminins
Abstract
The present disclosure describes methods of maintaining the
phenotype of differentiated cells. Generally, the natural
environment of the body is replicated for the differentiated cell.
The differentiated cell is plated on a cell culture substrate
comprising a laminin, such as laminin-521 or laminin-511. The
substrate may also contain a cadherin. This maintains the phenotype
of the differentiated cell.
Inventors: |
Tryggvason; Karl (Singapore,
SG), Tryggvason; Karl Kristian (Stockholm,
SE), Domogatskaya; Anna (Ronninge, SE) |
Applicant: |
Name |
City |
State |
Country |
Type |
BioLamina AB |
Stockholm |
N/A |
SE |
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Assignee: |
BIOLAMINA AB (Sundyberg,
SE)
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Family
ID: |
1000005891476 |
Appl.
No.: |
15/351,124 |
Filed: |
November 14, 2016 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20170067016 A1 |
Mar 9, 2017 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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13866177 |
Nov 22, 2016 |
9499794 |
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61754784 |
Jan 21, 2013 |
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61716005 |
Oct 19, 2012 |
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61636293 |
Apr 20, 2012 |
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61636211 |
Apr 20, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
5/0676 (20130101); C12N 5/0068 (20130101); C12N
2533/52 (20130101); C12N 2501/998 (20130101) |
Current International
Class: |
C12N
5/00 (20060101); C12N 5/071 (20100101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Meenal Banerjee, Ismo Virtanen, Jaan Palgi, Olle Korsgren and Timo
Otonkoski, Proliferation and plasticity of human beta cells on
physiologically occurring laminin isoforms, 2012, Molecular and
Cellular Endocrinology, vol. 355. pp. 78-86. (Year: 2012). cited by
examiner .
Extended European Search Report of European Application No.
17194017.4, dated Nov. 2, 2017. cited by applicant .
Banerjee Meenal et al., "Proliferation and plasticity of human beta
cells on physiologically occurring laminin isoforms", Molecular and
Cellular Endocrinology, vol. 355, No. 1, Jan. 31, 2012 (Jan. 31,
2012), pp. 78-86, XP002711864, ISSN: 0303-7207 p. 83, left-hand
column, last paragraph--right-hand column, last paragraph; figure
5. cited by applicant .
Zukowska-Grojec Zofia et al: "Neuropeptide Y. A novel angiogenic
factor from the sympathetic nerves and endothelium", Circulation
Research, Grune and Stratton, Baltimore, US, vol. 83, No. 2, Jul.
27, 1998 (Jul. 27, 1998), pp. 187-195, XP002958471, ISSN: 0009-7330
p. 188, left-hand column, last paragraph. cited by applicant .
Michael Zeisberg et al: "De-differentiation of primary human
hepatocytes depehds on the composition of specialized liver
basement membrane", Molecular and Cellular Biochemistry, Kluwer
Academic Publishers, BO, vol. 283, No. 1-2, Feb. 1, 2006 (Feb. 1,
2006), pp. 181-189, XP019289028, ISSN: 1573-4919 p. 182, left-hand
column, last paragraph--p. 183, left-hand column, paragraph 1.
cited by applicant .
Cotton M et al, "Transferrin-Polycation-Mediated Introduction of
DNA into Human Leukemic Ceels: Stimulation by Agents that Affect
the Survival of Transfected DNA or Modulate Transferring Receptor
Levels," PNAS, National Academy of Sciences, Jun. 1, 1990, pp.
4033-4037, vol. 87, US. cited by applicant.
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Primary Examiner: Berke-Schlessel; David W
Assistant Examiner: Clarke; Trent R
Attorney, Agent or Firm: Fay Sharpe LLP
Parent Case Text
This application is a continuation of U.S. patent application Ser.
No. 13/866,177, filed on Apr. 19, 2013, now U.S. Pat. No.
9,499,794, which claims priority to U.S. Provisional Patent
Application Ser. No. 61/716,005, filed on Oct. 19, 2012; to U.S.
Provisional Patent Application Ser. No. 61/754,784, filed on Jan.
21, 2013; to U.S. Provisional Patent Application Ser. No.
61/636,293, filed on Apr. 20, 2012; and to U.S. Provisional Patent
Application Ser. No. 61/636,211, filed on Apr. 20, 2012. The
disclosure of each of these applications is hereby fully
incorporated by reference.
Claims
The invention claimed is:
1. A method for maintaining the phenotype of a differentiated cell
in a cell culture, comprising: forming a cell culture substrate as
a layer on a surface, the layer comprising a laminin, wherein the
laminin is an intact protein or a protein fragment; plating the
differentiated cell directly on the cell culture substrate; and
culturing the plated differentiated cell on the layer; wherein the
laminin is laminin-521, and wherein the differentiated cell is a
pancreatic islet cell.
2. The method of claim 1, wherein the laminin is an effective
recombinant laminin.
3. The method of claim 1, further including applying a cell culture
medium to the differentiated cell.
4. The method of claim 3, wherein the cell culture medium has an
albumin concentration of at least 0.3 mM.
5. The method according to claim 1, wherein the phenotype is
maintained for at least 3 weeks in culture.
6. The method according to claim 5, wherein the maintained
pancreatic islet cell phenotype comprises insulin expression.
7. The method according to claim 6, wherein the insulin expression
is evidenced by positive staining for C-peptide.
8. The method according to claim 6, wherein the maintained
pancreatic islet cell phenotype comprises capacity for
proliferation.
9. The method according to claim 7, wherein the maintained
pancreatic islet cell phenotype comprises capacity for
proliferation.
Description
BACKGROUND
A stem cell is an undifferentiated cell from which specialized
cells are subsequently derived. Examples of stem cells in the human
body include pluripotent stem cells, embryonic stem cells, adult
stem cells, fetal stem cells, and amniotic stem cells. Embryonic
stem cells possess extensive self-renewal capacity and pluripotency
with the potential to differentiate into cells of all three germ
layers.
Totipotency refers to a cell that has the ability to differentiate
into any cell in the body, including extraembryonic tissue.
Pluripotency refers to a cell that has the potential to
differentiate into cells of all three germ layers. Pluripotent
cells however cannot form extraembryonic tissue, as a totipotent
cell can. Multipotency refers to a cell that can differentiate into
cells of limited lineage. For example, a hematopoietic stem cell
can differentiate into several types of blood cells, but cannot
differentiate into a brain cell.
The process by which a stem cell changes into a more specialized
cell is referred to as differentiation. For example, some
differentiated cells include endothelial cells, which are derived
from endothelial stem cells.
The process by which a specialized cell reverts back to a higher
degree of potency (i.e. to an earlier developmental stage) is
referred to as dedifferentiation. In particular, cells in a cell
culture can lose properties they originally had, such as protein
expression or shape. It would be desirable to reduce the rate of
dedifferentiation, or in other words to maintain the phenotype of
differentiated cells in a cell culture.
BRIEF DESCRIPTION
Disclosed herein are methods for maintaining the phenotype of
differentiated cells in a cell culture.
Described herein are methods for maintaining the phenotype of a
differentiated cell, comprising: plating the differentiated cell on
a cell culture substrate comprising a laminin, wherein the laminin
is an intact protein or a protein fragment.
The differentiated cell can be an endothelial cell, a
cardiomyocyte, a dopamine producing cell, a hepatocyte, or a
pancreatic beta cell.
The laminin may be laminin-521 or laminin-511, or an effective
recombinant laminin.
The cell culture substrate may further comprise a cadherin.
Sometimes, the cadherin is e-cadherin. The weight ratio of the
laminin to the cadherin can be from about 5:1 to about 15:1, or
from about 5:1 to about 10:1. In particular embodiments, the
laminin is laminin-521 and the cadherin is e-cadherin. In other
embodiments, the cell culture substrate consists of the laminin and
the cadherin. Generally, the cell culture substrate does not
contain any differentiation inhibitors, feeder cells,
differentiation inductors, or apoptosis inhibitors.
The method may further include applying a cell culture medium to
the first stem cell. In specific embodiments, the cell culture
medium has an albumin concentration of at least 0.3 mM.
These and other non-limiting characteristics of the disclosure are
more particularly disclosed below.
BRIEF DESCRIPTION OF THE DRAWINGS
The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
The following is a brief description of the drawings, which are
presented for the purposes of illustrating the exemplary
embodiments disclosed herein and not for the purposes of limiting
the same.
FIG. 1 is a rotary shadowing electron microscopy picture of a
recombinant laminin molecule.
FIG. 2 shows the structural motifs of laminin .alpha., .beta., and
.gamma. chains. The N-terminal, internal, and C-terminal globular
domains are depicted as white ovals. The coiled-coil forming
domains (I and II) are shown as white rectangles. The rod-like
structures (domains V, IIIb, and IIIa) are depicted as grey
rectangles.
FIG. 3 is a photomicrograph of human umbilical vein endothelial
cells (HUVECs) grown on a fibronectin (FNE) substrate, 10.times.
magnification, after 5 passages, with expression of von Willebrand
factor (vWF), f-actin, and DAPI.
FIG. 4 is a photomicrograph of HUVECs grown on a LN-521 substrate,
10.times. magnification, after 5 passages, with expression of vWF,
f-actin, and DAPI.
FIG. 5 is a photomicrograph of HUVECs grown on a LN-521 substrate,
10.times. magnification, after 7 passages, with expression of vWF,
f-actin, and DAPI.
FIG. 6 is a photomicrograph of HUVECs grown on a LN-411/511
substrate, 10.times. magnification, after 5 passages, with
expression of vWF, f-actin, and DAPI.
FIG. 7 is a photomicrograph of HUVECs grown on a LN-511 substrate,
10.times. magnification, after 5 passages, with expression of vWF,
f-actin, and DAPI.
FIG. 8 is graph of RNA Gene Expression showing relatively low Acta2
gene expression (negative marker) (the five leftmost columns) and
high vWF gene expression (positive marker) (the five rightmost
columns) relative to a control (Fibronectin) when cells are grown
on a LN-511 substrate.
FIG. 9 is a graph of quantified percentage of vWF-positive HUVECs
within a population after a long-term culture of HUVECs on human
recombinant laminin-521.
FIG. 10 is graph of quantified percentage of vWF-positive HUVECs
within a population after a long-term culture of HUVECs on human
recombinant Fibronectin.
FIG. 11 is proliferation curve showing the proliferation of HUVECs
on different substrate coatings, dependent of days in culture. The
LN-521 line always has the greatest value. The LN-411/511 and
LN-511 lines essentially overlap until .about.115 days, at which
point the LN-511 line is greater. The FNE line has the lowest value
until .about.80 days, when it then crosses over to have the
second-highest value.
FIG. 12 is a phase-contrast micrograph of mouse pancreatic
insulin-producing islet beta cells plated on a surface coated with
LN-521, 10.times. magnification, 3 week culture.
FIG. 13 is a phase-contrast micrograph of mouse pancreatic
insulin-producing islet beta cells plated on an uncoated surface,
10.times. magnification, 3 week culture.
FIG. 14 is a phase-contrast micrograph of mouse pancreatic
insulin-producing islet beta cells plated on a surface coated with
LN-411, 10.times. magnification, 3 week culture.
FIG. 15 is a phase-contrast micrograph of mouse pancreatic
insulin-producing islet beta cells plated on a surface coated with
LN-511, 10.times. magnification, 3 week culture.
FIG. 16 is a phase-contrast micrograph of mouse pancreatic
insulin-producing islet beta cells plated on a surface coated with
LN-111, 10.times. magnification, 3 week culture.
FIG. 17 is a phase-contrast micrograph of mouse pancreatic
insulin-producing islet beta cells plated on a surface coated with
human recombinant LN-521 at (a) 10.times. magnification and (b)
40.times. magnification.
FIG. 18 is a phase-contrast micrograph of mouse pancreatic
insulin-producing islet beta cells plated on a surface coated with
human recombinant LN-111 at (a) 10.times. magnification and (b)
40.times. magnification.
FIG. 19 is a phase-contrast micrograph of mouse pancreatic
insulin-producing islet beta cells plated on a surface coated with
human recombinant LN-521 for 3-4 weeks at 10.times. magnification
and subsequently (a) stained positively for C-peptide (green) and
(b) stained positively for C-peptide (green) and Hoechst
(blue).
FIG. 20 is a phase-contrast micrograph of mouse pancreatic
insulin-producing islet beta cells plated on a surface coated with
human recombinant LN-521 for 3-4 weeks at 20.times. magnification
and subsequently (a) stained positively for EdU (green) in nuclei
of proliferated cells and (b) stained positively for Edu (green)
co-localized with phase contrast photograph of islet.
FIG. 21 is a phase-contrast micrograph of mouse pancreatic
insulin-producing islet beta cells plated on a surface coated with
human recombinant LN-521 for 3-4 weeks at 20.times. magnification
and subsequently (a) stained positively for EdU (green) in nuclei
of proliferated cells and (b) stained positively for Edu (green)
co-localized with Hoechst (blue).
DETAILED DESCRIPTION
A more complete understanding of the compositions and methods
disclosed herein can be obtained by reference to the accompanying
drawings. These figures are merely schematic representations based
on convenience and the ease of demonstrating the present
disclosure, and are, therefore, not intended to define or limit the
scope of the exemplary embodiments.
Although specific terms are used in the following description for
the sake of clarity, these terms are intended to refer only to the
particular structure of the embodiments selected for illustration
in the drawings, and are not intended to define or limit the scope
of the disclosure. In the drawings and the following description
below, it is to be understood that like numeric designations refer
to components of like function.
All publications, patents, and patent applications discussed herein
are hereby incorporated by reference in their entirety.
Unless otherwise stated, the techniques utilized in this
application may be found in any of several well-known references
such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al.,
1989, Cold Spring Harbor Laboratory Press), Gene Expression
Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel,
1991. Academic Press, San Diego, Calif.), "Guide to Protein
Purification" in Methods in Enzymology (M. P. Deutshcer, ed.,
(1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and
Applications (Innis, et al. 1990. Academic Press, San Diego,
Calif.), Culture of Animal Cells: A Manual of Basic Technique,
Second Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), Gene
Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray,
The Humana Press Inc., Clifton, N.J.), or the Ambion 1998 Catalog
(Ambion, Austin, Tex.).
The methods of the present disclosure are generally related to
maintaining the phenotype of differentiated cells. The term
"phenotype" here refers to the cell's observable characteristics
and properties. These include such things as the cell's morphology,
biochemical or physiological properties, etc. It is desirable to
maintain the cell's phenotype.
It is contemplated that any kind of differentiated cell can be
maintained with the methods of the present disclosure. Examples of
differentiated cells include endothelial cells, cardiomyocytes,
dopamine producing cells, hepatocytes, and pancreatic beta cells,
though of course other differentiated cells are contemplated.
Generally speaking, the present disclosure creates a natural
environment for the differentiated cell using laminins that are
close to the differentiated cell in the body.
The methods of the present disclosure also relate to improving the
transfection efficiency of primary cells and/or survival rate of
transfected cells. The primary cells are plated on a substrate
containing a laminin, wherein the laminin is an intact protein or a
protein fragment. The primary cells are then transfected with a
vector, and the transfected cells are cultured on the
substrate.
The term "primary cells" refers in the art to cells which are taken
directly from a subject. Such cells generally are not immortal, and
have a limited lifespan, or in other words they stop dividing
though they retain viability. Exemplary primary cells include
hepatocytes, adipocytes, podocytes, chondrocytes, melanocytes,
keratinocytes, and laminins. Primary cells are also differentiated
cells.
Differentiated cells typically require two things to survive and
reproduce: (1) a substrate or coating that provides a structural
support for the cell; and (2) a cell culture medium to provide
nutrition to the cell. The substrate or coating (1) is typically
formed as a layer in a container, for example a petri dish or in
the well of a multi-well plate. It is particularly contemplated
that the cell culture substrate on which the differentiated cell is
plated comprises a laminin and a cadherin.
Laminins are a family of heterotrimeric glycoproteins that reside
primarily in the basal lamina. They function via binding
interactions with neighboring cell receptors on the one side, and
by binding to other laminin molecules or other matrix proteins such
as collagens, nidogens or proteoglycans. The laminin molecules are
also important signaling molecules that can strongly influence
cellular behavior and function. Laminins are important in both
maintaining cell/tissue phenotype, as well as in promoting cell
growth and differentiation in tissue repair and development.
Laminins are large, multi-domain proteins, with a common structural
organization. The laminin molecule integrates various matrix and
cell interactive functions into one molecule.
A laminin protein molecule comprises one .alpha.-chain subunit, one
.beta.-chain subunit, and one .gamma.-chain subunit, all joined
together in a trimer through a coiled-coil domain. FIG. 1 depicts
the resulting structure of the laminin molecule. The twelve known
laminin subunit chains can form at least 15 trimeric laminin types
in native tissues. Within the trimeric laminin structures are
identifiable domains that possess binding activity towards other
laminin and basal lamina molecules, and membrane-bound receptors.
FIG. 2 shows the three laminin chain subunits separately. For
example, domains VI, IVb, and IVa form globular structures, and
domains V, IIIb, and IIIa (which contain cysteine-rich EGF-like
elements) form rod-like structures. Domains I and II of the three
chains participate in the formation of a triple-stranded
coiled-coil structure (the long arm).
There exist five different alpha chains, three beta chains and
three gamma chains that in human tissues have been found in at
least fifteen different combinations. These molecules are termed
laminin-1 to laminin-15 based on their historical discovery, but an
alternative nomenclature describes the isoforms based on their
chain composition, e.g. laminin-111 (laminin-1) that contains
alpha-1, beta-1 and gamma-1 chains. Four structurally defined
family groups of laminins have been identified. The first group of
five identified laminin molecules all share the .beta.1 and
.gamma.1 chains, and vary by their .alpha.-chain composition
(.alpha.1 to .alpha.5 chain). The second group of five identified
laminin molecules, including laminin-521, all share the .beta.2 and
.gamma.1 chain, and again vary by their .alpha.-chain composition.
The third group of identified laminin molecules has one identified
member, laminin-332, with a chain composition of
.alpha.3.beta.3.gamma.2. The fourth group of identified laminin
molecules has one identified member, laminin-213, with the newly
identified .gamma.3 chain (.alpha.2.beta.1.gamma.3).
Generally, the cell culture substrate may contain any effective
laminin, wherein the effectiveness is determined by whether
differentiated cells can survive upon the substrate. It is
specifically contemplated that the substrate contains only one
particular laminin, though other ingredients are also present in
the substrate. In specific embodiments, the laminin is laminin-521
(LN-521) or laminin-511 (LN-511).
The term "laminin-521" refers to the protein formed by joining
.alpha.5, .beta.2 and .gamma.1 chains together. The term
"laminin-511" refers to the protein formed by joining .alpha.5,
.beta.1 and .gamma.1 chains together. These terms should be
construed as encompassing both the recombinant laminin and
heterotrimeric laminin from naturally occurring sources. The term
"recombinant" indicates that the protein is artificially produced
in cells that do not normally express such proteins.
The laminin can be an intact protein or a protein fragment. The
term "intact" refers to the protein being composed of all of the
domains of the .alpha.-chain, .beta.-chain, and .gamma.-chain, with
the three chains being joined together to form the heterotrimeric
structure. The protein is not broken down into separate chains,
fragments, or functional domains. The term "chain" refers to the
entirety of the alpha, beta, or gamma chain of the laminin protein.
The term "fragment" refers to any protein fragment which contains
one, two, or three functional domains that possesses binding
activity to another molecule or receptor. However, a chain should
not be considered a fragment because each chain possesses more than
three such domains. Similarly, an intact laminin protein should not
be considered a fragment. Examples of functional domains include
Domains I, II, III, IV, V, VI, and the G domain.
The average contact area and spreading homogeneity is much larger
for cells cultured on laminin-511 compared to other available
substrata.
In particular, it is noted that the pancreatic insulin-producing
islets are naturally in the shape of a three-dimensional sphere.
However, a petri dish typically only provides two dimensions for
growth, which means that it is difficult to expand the islets using
a mechanical split. Beta cells within the islets form
syncytium-like structures, and beta cells will respond
simultaneously to external signals. When cultured as single cells,
though, beta cells lose this natural function of simultaneous
response.
The cell culture substrate also comprises a cadherin. Cadherins are
a class of type-1 transmembrane proteins that play important roles
in cell adhesion, ensuring that cells within tissues are bound
together. They are dependent on calcium (Ca.sup.2+) ions to
function. Cadherins are also known as desmogleins and desmocollins.
Structurally, cadherins contain extracellular Ca.sup.2+-binding
domains. In particular embodiments, the cadherin used in the cell
culture substrate is epithelial cadherin or e-cadherin.
The weight ratio of the laminin to the cadherin may be from about
5:1 to about 15:1, or from about 5:1 to about 10:1. In particular
embodiments, the cell culture substrate consists of the laminin and
the cadherin. In other specific embodiments, the laminin is
laminin-521 and the cadherin is e-cadherin.
The cell culture substrate is used in combination with a cell
culture medium. The cell culture medium of the present disclosure
is particularly suitable for being used with a substrate that
contains laminin-521 and/or laminin-511. These laminins activate
.alpha.6.beta.1 integrins, which in turn leads to activation of the
PI3K/Akt pathway. This increases the pluripotency, self-renewal,
and/or proliferation of the differentiated cells. It is
contemplated that the substrate may consist of laminin-521 or
laminin-511, either intact, as separate chains, or as fragments
thereof. Recombinant laminin-521 and recombinant laminin-511 are
commercially available; see for example U.S. Pat. No. 8,415,156,
which provides amino acid sequences and DNA sequences for LN-521,
and the entirety of which is incorporated by reference herein. Many
different molecules can activate the PI3K/Akt pathway, though with
different efficiencies. For example, TGF beta 1 and bFGF activate
this pathway. The use of laminin-521 and/or laminin-511 allows the
quantity of such molecules to be reduced in the cell culture
medium. Laminin-521 conveys the highest dose of signal via
.alpha.6.beta.1 integrin, activating the PI3K/Akt pathway. The use
of laminin-521 allows for single-cell suspension passaging without
the addition of cell-detrimental rho-kinase (ROCK) inhibitor to
increase cell survival after single-cell enzymatic
dissociation.
Typically, cell culture media include a large number and a large
amount of various growth factors and cytokines to inhibit
differentiation and improve proliferation. One advantage of the
cell culture medium of the present disclosure is that it does not
contain as many growth factors or cytokines, or such high
amounts.
Very generally, the cell culture medium of the present disclosure
requires lower amounts of basic fibroblast growth factor (bFGF)
than typically used. It is contemplated that the cell culture
medium may comprise from greater than zero to 3.9 nanograms per
milliliter (ng/mL) of bFGF. The bFGF is human bFGF so that the cell
culture medium is totally human and defined. In some more specific
embodiments, the cell culture medium may comprise 3.5 or lower
ng/mL of bFGF. In other embodiments, the cell culture medium may
comprise from 0.5 to 3.5 ng/mL of bFGF. In some embodiments, the
cell culture medium may have zero bFGF, i.e. no bFGF is
present.
Generally, the cell culture medium includes a liquid phase in which
at least one inorganic salt, at least one trace mineral, at least
one energy substrate, at least one lipid, at least one amino acid,
at least one vitamin, and at least one growth factor (besides bFGF)
are dissolved. Table 1 below includes a list of various such
ingredients which may be present in the cell culture medium of the
present disclosure, and the minimum and maximum concentrations if
the ingredient is present. The values are presented in scientific
notation. For example, "4.1E-01" should be interpreted as
4.1.times.10.sup.-01.
TABLE-US-00001 TABLE 1 molar Min. Max. Min. Max. mass Conc. Conc.
Conc. Conc. Ingredient (g/mol) (mM) (mM) (ng/mL) (ng/mL) INORGANIC
SALTS Calcium chloride 110.98 4.1E-01 1.6E+00 4.6E+04 1.8E+05
(Anhydrous) HEPES 238.3 5.9E+00 1.8E+01 1.4E+06 4.2E+06 Lithium
Chloride (LiCl) 42.39 4.9E-01 1.5E+00 2.1E+04 6.2E+04 Magnesium
chloride 95.21 1.2E-01 3.6E-01 1.1E+04 3.4E+04 (Anhydrous)
Magnesium Sulfate 120.37 1.6E-01 4.8E-01 1.9E+04 5.8E+04
(MgSO.sub.4) Potassium chloride (KCl) 74.55 1.6E+00 4.9E+00 1.2E+05
3.6E+05 Sodium bicarbonate 84.01 9.0E+00 4.4E+01 7.6E+05 3.7E+06
(NaHCO.sub.3) Sodium chloride (NaCl) 58.44 4.7E+01 1.4E+02 2.8E+06
8.3E+06 Sodium phosphate, 141.96 2.0E-01 5.9E-01 2.8E+04 8.3E+04
dibasic (Anhydrous) Sodium phosphate, 137.99 1.8E-01 5.3E-01
2.4E+04 7.3E+04 monobasic monohydrate (NaH.sub.2PO.sub.4--H.sub.2O)
TRACE MINERALS Ferric Nitrate (Fe(NO.sub.3).sub.3--9H.sub.2O) 404
4.9E-05 1.9E-04 2.0E+01 7.5E+01 Ferrous sulfate 278.01 5.9E-04
1.8E-03 1.6E+02 4.9E+02 heptahydrate (FeSO.sub.4--7H.sub.2O)
Copper(II) sulfate 249.69 2.0E-06 8.0E-06 5.1E-01 2.0E+00
pentahydrate (CuSO.sub.4--5H.sub.2O) Zinc sulfate heptahydrate
287.56 5.9E-04 1.8E-03 1.7E+02 5.1E+02 (ZnSO.sub.4--7H.sub.2O)
Ammonium Metavanadate 116.98 5.5E-06 1.6E-05 6.4E-01 1.9E+00
NH.sub.4VO.sub.3 Manganese Sulfate 169.02 9.9E-07 3.0E-06 1.7E-01
5.0E-01 monohydrate (MnSO.sub.4--H.sub.2O) NiSO.sub.4--6H.sub.2O
262.85 4.9E-07 1.5E-06 1.3E-01 3.8E-01 Selenium 78.96 8.9E-05
2.7E-04 7.0E+00 2.1E+01 Sodium Meta Silicate 284.2 4.8E-04 1.4E-03
1.4E+02 4.1E+02 Na.sub.2SiO.sub.3--9H.sub.2O SnCl.sub.2 189.62
6.2E-07 1.9E-06 1.2E-01 3.5E-01 Molybdic Acid, Ammonium 1235.86
9.9E-07 3.0E-06 1.2E+00 3.7E+00 salt CdCl.sub.2 183.32 6.1E-06
1.8E-05 1.1E+00 3.4E+00 CrCl.sub.3 158.36 9.9E-07 3.0E-06 1.6E-01
4.7E-01 AgNO.sub.3 169.87 4.9E-07 1.5E-06 8.3E-02 2.5E-01
AlCl.sub.3--6H.sub.2O 241.43 2.4E-06 7.3E-06 5.9E-01 1.8E+00 Barium
Acetate 255.42 4.9E-06 1.5E-05 1.3E+00 3.8E+00
(Ba(C.sub.2H.sub.3O.sub.2).sub.2) CoCl.sub.2--6H.sub.2O 237.93
4.9E-06 1.5E-05 1.2E+00 3.5E+00 GeO.sub.2 104.64 2.5E-06 7.5E-06
2.6E-01 7.8E-01 KBr 119 4.9E-07 1.5E-06 5.9E-02 1.8E-01 Kl 166
5.0E-07 1.5E-06 8.3E-02 2.5E-01 NaF 41.99 4.9E-05 1.5E-04 2.1E+00
6.2E+00 RbCl 120.92 4.9E-06 1.5E-05 5.9E-01 1.8E+00
ZrOCl.sub.2--8H.sub.2O 178.13 4.9E-06 1.5E-05 8.7E-01 2.6E+00
ENERGY SUBSTRATES D-Glucose 180.16 6.9E+00 2.1E+01 1.2E+06 3.7E+06
Sodium Pyruvate 110.04 2.0E-01 5.9E-01 2.2E+04 6.5E+04 LIPIDS
Linoleic Acid 280.45 9.4E-05 2.8E-04 2.6E+01 7.9E+01 Lipoic Acid
206.33 2.0E-04 7.8E-04 4.1E+01 1.6E+02 Arachidonic Acid 304.47
6.5E-06 1.9E-05 2.0E+00 5.9E+00 Cholesterol 386.65 5.6E-04 1.7E-03
2.2E+02 6.5E+02 DL-alpha tocopherol- 472.74 1.5E-04 4.4E-04 6.9E+01
2.1E+02 acetate Linolenic Acid 278.43 3.5E-05 1.0E-04 9.7E+00
2.9E+01 Myristic Acid 228.37 4.3E-05 1.3E-04 9.8E+00 2.9E+01 Oleic
Acid 282.46 3.5E-05 1.0E-04 9.8E+00 2.9E+01 Palmitic Acid 256.42
3.8E-05 1.1E-04 9.8E+00 2.9E+01 Palmitoleic acid 254.408 3.9E-05
1.2E-04 9.8E+00 2.9E+01 Stearic Acid 284.48 3.4E-05 1.0E-04 9.8E+00
2.9E+01 AMINO ACIDS L-Alanine 89.09 2.5E-02 2.1E-01 2.2E+03 1.8E+04
L-Arginine hydrochloride 147.2 2.7E-01 1.5E+00 4.0E+04 2.2E+05
L-Asparagine-H.sub.2O 150.13 5.0E-02 2.1E-01 7.5E+03 3.1E+04
L-Aspartic acid 133.1 2.5E-02 2.1E-01 3.3E+03 2.7E+04
L-Cysteine-HCl--H.sub.2O 175.63 3.9E-02 1.2E-01 6.9E+03 2.1E+04
L-Cystine dihydrochloride 313.22 3.9E-02 1.2E-01 1.2E+04 3.7E+04
L-Glutamic acid 147.13 2.5E-02 2.1E-01 3.7E+03 3.0E+04 L-Glutamine
146.15 1.5E+00 4.4E+00 2.1E+05 6.4E+05 Glycine 75.07 1.5E-01
4.4E-01 1.1E+04 3.3E+04 L-Histidine 209.63 5.9E-02 1.8E-01 1.2E+04
3.7E+04 monohydrochloride monohydrate L-Isoleucine 131.17 1.6E-01
4.9E-01 2.1E+04 6.4E+04 L-Leucine 131.17 1.8E-01 5.3E-01 2.3E+04
7.0E+04 L-Lysine hydrochloride 182.65 2.0E-01 5.9E-01 3.6E+04
1.1E+05 L-Methionine 149.21 4.5E-02 1.4E-01 6.8E+03 2.0E+04
L-Phenylalanine 165.19 8.5E-02 2.5E-01 1.4E+04 4.2E+04 L-Proline
115.13 1.1E-01 3.2E-01 1.2E+04 3.7E+04 L-Serine 105.09 1.5E-01
4.4E-01 1.5E+04 4.6E+04 L-Threonine 119.12 1.8E-01 5.3E-01 2.1E+04
6.3E+04 L-Tryptophan 204.23 1.7E-02 5.2E-02 3.5E+03 1.1E+04
L-Tyrosine disodium salt 225.15 8.4E-02 3.7E-01 1.9E+04 8.4E+04
hydrate L-Valine 117.15 1.8E-01 5.3E-01 2.1E+04 6.2E+04 VITAMINS
Ascorbic acid 176.12 1.3E-01 3.8E-01 2.2E+04 6.7E+04 Biotin 244.31
5.6E-06 1.7E-05 1.4E+00 4.1E+00 B.sub.12 1355.37 2.0E-04 5.9E-04
2.7E+02 8.0E+02 Choline chloride 139.62 2.5E-02 7.5E-02 3.5E+03
1.1E+04 D-Calcium pantothenate 238.27 1.8E-03 1.4E-02 4.4E+02
3.4E+03 Folic acid 441.4 2.4E-03 7.1E-03 1.0E+03 3.1E+03 i-Inositol
180.16 2.7E-02 1.1E-01 4.9E+03 1.9E+04 Niacinamide 122.12 6.5E-03
2.0E-02 7.9E+02 2.4E+03 Pyridoxine hydrochloride 205.64 3.8E-03
1.1E-02 7.8E+02 2.4E+03 Riboflavin 376.36 2.3E-04 6.8E-04 8.6E+01
2.6E+02 Thiamine hydrochloride 337.27 3.3E-03 3.6E-02 1.1E+03
1.2E+04 GROWTH FACTORS/PROTEINS GABA 103.12 0 1.5E+00 0 1.5E+05
Pipecolic Acid 129 0 1.5E-03 0 1.9E+02 bFGF 18000 0 2.17E-07 0
3.9E+00 TGF beta 1 25000 0 3.5E-08 0 8.8E-01 Human Insulin 5808 0
5.9E-03 0 3.4E+04 Human Holo-Transferrin 78500 0 2.1E-04 0 1.6E+04
Human Serum Albumin 67000 0 2.9E-01 0 2.0E+07 Glutathione (reduced)
307.32 0 9.6E-03 0 2.9E+03 OTHER COMPONENTS Hypoxanthine Na 136.11
5.9E-03 2.6E-02 8.0E+02 3.6E+03 Phenol red 354.38 8.5E-03 2.5E-02
3.0E+03 9.0E+03 Putrescine-2HCl 161.07 2.0E-04 5.9E-04 3.2E+01
9.5E+01 Thymidine 242.229 5.9E-04 1.8E-03 1.4E+02 4.3E+02
2-mercaptoethanol 78.13 4.9E-02 1.5E-01 3.8E+03 1.1E+04 Pluronic
F-68 8400 1.2E-02 3.5E-02 9.8E+04 2.9E+05 Tween 80 1310 1.6E-04
4.9E-04 2.2E+02 6.5E+02
The liquid phase of the cell culture medium may be water, serum, or
albumin.
Many of the ingredients or components listed above in Table 1 are
not necessary, or can be used in lower concentrations.
It is contemplated that the cell culture medium may contain insulin
or an insulin substitute. Similarly, the cell culture medium may
contain transferrin or a transferrin substitute. However, in more
specific embodiments, it is contemplated that the cell culture
medium may not (1) insulin or insulin substitute, or (2)
transferrin or transferrin substitute, or any combination of these
two components.
It should be noted that other cell culture mediums may contain
growth factors such as interleukin-1 beta (IL-1.beta. or
catabolin), interleukin-6 (IL6), or pigment epithelium derived
factor (PEDF). Such growth factors are not present in the cell
culture medium of the present disclosure.
One specific formula for a cell culture medium is provided in Table
2:
TABLE-US-00002 TABLE 2 Ingredient Amount Unit bFGF 0.39 microgram
(.mu.g) Albumin 1.34 milligram (mg) Insulin 2 mg Lithium Chloride
4.23 mg GABA 0.01 mg TGF beta 1 0.06 .mu.g Pipecolic acid 0.013 mg
L-glutamine 2.92 grams MEM non-essential amino acid solution 1 mL
DMEM/F12 100 mL
In this regard, MEM non-essential amino acid solution is typically
provided in a 100.times. concentrate. The MEM of Table 2 is used
after dilution back to 1.times., and contains the following amino
acids in the following concentration listed in Table 3:
TABLE-US-00003 TABLE 3 MEM Concentration Amino Acids (ng/mL)
Glycine 7.50E+03 L-Alanine 8.90E+03 L-Asparagine 1.32E+04
L-Aspartic acid 1.33E+04 L-Proline 1.15E+04 L-Serine 1.05E+04
DMEM/F12 contains the following ingredients listed in Table 4:
TABLE-US-00004 TABLE 4 Concentration DMEM/F12 Ingredients (ng/mL)
Glycine 187.5 L-Alanine 44.5 L-Arginine hydrochloride 1475
L-Asparagine-H.sub.2O 75 L-Aspartic acid 66.5 L-Cysteine
hydrochloride-H.sub.2O 175.6 L-Cystine 2HCl 312.9 L-Glutamic Acid
73.5 L-Glutamine 3650 L-Histidine hydrochloride-H.sub.2O 314.8
L-Isoleucine 544.7 L-Leucine 590.5 L-Lysine hydrochloride 912.5
L-Methionine 172.4 L-Phenylalanine 354.8 L-Proline 172.5 L-Serine
262.5 L-Threonine 534.5 L-Tryptophan 90.2 L-Tyrosine disodium salt
dihydrate 557.9 L-Valine 528.5 Biotin 0.035 Choline chloride 89.8
D-Calcium pantothenate 22.4 Folic Acid 26.5 Niacinamide 20.2
Pyridoxine hydrochloride 20 Riboflavin 2.19 Thiamine hydrochloride
21.7 Vitamin B.sub.12 6.8 i-Inositol 126 Calcium Chloride
(CaCl.sub.2) (anhyd.) 1166 Cupric sulfate (CuSO.sub.4--5H.sub.2O)
0.013 Ferric Nitrate (Fe(NO.sub.3).sub.3--9H.sub.2O) 0.5 Ferric
sulfate (FeSO.sub.4--7H.sub.2O) 4.17 Magnesium Chloride (anhydrous)
286.4 Magnesium Sulfate (MgSO.sub.4) (anhyd.) 488.4 Potassium
Chloride (KCl) 3118 Sodium Bicarbonate (NaHCO.sub.3) 24380 Sodium
Chloride (NaCl) 69955 Sodium Phosphate dibasic 710.2
(Na.sub.2HPO.sub.4) anhydrous Sodium Phosphate monobasic 625
(NaH.sub.2PO.sub.4--H.sub.2O) Zinc sulfate (ZnSO.sub.4--7H.sub.2O)
4.32 D-Glucose (Dextrose) 31510 Hypoxanthine Na 23.9 Linoleic Acid
0.42 Lipoic Acid 1.05 Phenol Red 81 Putrescine 2HCl 0.81 Sodium
Pyruvate 550 Thymidine 3.65
In particular, the cell culture medium may have an albumin
concentration of at least 0.3 millimolar (mM). It has been found
that a 2.times. increase in albumin concentration significantly
improved clonal survival of human embryonic stem cells on a
laminin-521/E-Cadherin matrix. Table 5 below provides a formulation
for a cell culture medium containing additional albumin.
In particular embodiments, the amount of human serum albumin (HSA)
can be varied from a concentration of 0.195 mM to 1 mM, including
from 0.3 mM to 1 mM or from 0.3 mM to about 0.4 mM. The amount of
bFGF can also be varied from 0 to about 105 ng/mL, or from 0 to 3.9
ng/mL, or from 0.5 ng/mL to 3.5 ng/mL. These two variations in the
amount of HSA and bFGF may occur independently or together.
TABLE-US-00005 TABLE 5 mTeSR1 formulation. molar mass Concentration
Concentration mTeSR1 Ingredient (g/mol) (ng/mL) (mM) INORGANIC
SALTS Calcium chloride (Anhydrous) 110.98 9.14E+04 8.24E-01 HEPES
238.3 2.81E+06 1.18E+01 Lithium Chloride (LiCl) 42.39 4.15E+04
9.80E-01 Magnesium chloride (Anhydrous) 95.21 2.26E+04 2.37E-01
Magnesium Sulfate (MgSO.sub.4) 120.37 3.84E+04 3.19E-01 Potassium
chloride (KCl) 74.55 2.43E+05 3.26E+00 Sodium bicarbonate
(NaHCO.sub.3) 84.01 1.51E+06 1.80E+01 Sodium chloride (NaCl) 58.44
5.53E+06 9.46E+01 Sodium phosphate, dibasic (Anhydrous) 141.96
5.56E+04 3.92E-01 Sodium phosphate, monobasic 137.99 4.90E+04
3.55E-01 monohydrate (NaH.sub.2PO.sub.4--H.sub.2O) TRACE MINERALS
Ferric Nitrate (Fe(NO.sub.3).sub.3--9H.sub.2O) 404 3.92E+01
9.71E-05 Ferrous sulfate heptahydrate (FeSO.sub.4--7H.sub.2O)
278.01 3.28E+02 1.18E-03 Copper(II) sulfate pentahydrate
(CuSO.sub.4--5H.sub.2O) 249.69 1.02E+00 4.08E-06 Zinc sulfate
heptahydrate (ZnSO.sub.4--7H.sub.2O) 287.56 3.39E+02 1.18E-03
Ammonium Metavanadate NH.sub.4VO.sub.3 116.98 1.28E+00 1.09E-05
Manganese Sulfate monohydrate (MnSO.sub.4--H.sub.2O) 169.02
3.33E-01 1.97E-06 NiSO.sub.4--6H.sub.2O 262.85 2.55E-01 9.70E-07
Selenium 78.96 1.40E+01 1.77E-04 Sodium Meta Silicate
Na.sub.2SiO.sub.3 9H.sub.2O 284.2 2.75E+02 9.66E-04 SnCl.sub.2
189.62 2.35E-01 1.24E-06 Molybdic Acid, Ammonium salt 1235.86
2.43E+00 1.97E-06 CdCl.sub.2 183.32 2.24E+00 1.22E-05 CrCl.sub.3
158.36 3.14E-01 1.98E-06 AgNO.sub.3 169.87 1.67E-01 9.81E-07
AlCl.sub.3 6H.sub.2O 241.43 1.18E+00 4.87E-06 Barium Acetate
(Ba(C.sub.2H.sub.3O.sub.2).sub.2) 255.42 2.50E+00 9.79E-06
CoCl.sub.2 6H.sub.2O 237.93 2.33E+00 9.81E-06 GeO.sub.2 104.64
5.20E-01 4.97E-06 KBr 119 1.18E-01 9.89E-07 Kl 166 1.66E-01
1.00E-06 NaF 41.99 4.13E+00 9.83E-05 RbCl 120.92 1.19E+00 9.81E-06
ZrOCl.sub.2 8H.sub.2O 178.13 1.75E+00 9.80E-06 ENERGY SUBSTRATES
D-Glucose 180.16 2.47E+06 1.37E+01 Sodium Pyruvate 110.04 4.31E+04
3.92E-01 LIPIDS Linoleic Acid 280.45 5.27E+01 1.88E-04 Lipoic Acid
206.33 8.25E+01 4.00E-04 Arachidonic Acid 304.47 3.93E+00 1.29E-05
Cholesterol 386.65 4.33E+02 1.12E-03 DL-alpha tocopherol-acetate
472.74 1.37E+02 2.90E-04 Linolenic Acid 278.43 1.95E+01 6.99E-05
Myristic Acid 228.37 1.96E+01 8.59E-05 Oleic Acid 282.46 1.96E+01
6.94E-05 Palmitic Acid 256.42 1.96E+01 7.65E-05 Palmitoleic acid
254.408 1.96E+01 7.71E-05 Stearic Acid 284.48 1.96E+01 6.89E-05
AMINO ACIDS L-Alanine 89.09 1.22E+04 1.37E-01 L-Arginine
hydrochloride 147.2 8.07E+04 5.48E-01 L-Asparagine-H.sub.2O 150.13
2.06E+04 1.37E-01 L-Aspartic acid 133.1 1.82E+04 1.37E-01
L-Cysteine-HCl--H.sub.2O 175.63 1.38E+04 7.83E-02 L-Cystine
dihydrochloride 313.22 2.45E+04 7.83E-02 L-Glutamic acid 147.13
2.02E+04 1.37E-01 L-Glutamine 146.15 4.30E+05 2.94E+00 Glycine
75.07 2.21E+04 2.94E-01 L-Histidine monohydrochloride 209.63
2.47E+04 1.18E-01 monohydrate L-Isoleucine 131.17 4.28E+04 3.26E-01
L-Leucine 131.17 4.64E+04 3.54E-01 L-Lysine hydrochloride 182.65
7.14E+04 3.91E-01 L-Methionine 149.21 1.35E+04 9.06E-02
L-Phenylalanine 165.19 2.79E+04 1.69E-01 L-Proline 115.13 2.49E+04
2.16E-01 L-Serine 105.09 3.09E+04 2.94E-01 L-Threonine 119.12
4.19E+04 3.52E-01 L-Tryptophan 204.23 7.07E+03 3.46E-02 L-Tyrosine
disodium salt hydrate 225.15 3.78E+04 1.68E-01 L-Valine 117.15
4.16E+04 3.55E-01 VITAMINS Ascorbic acid 176.12 4.46E+04 2.53E-01
Biotin 244.31 2.74E+00 1.12E-05 B12 1355.37 5.34E+02 3.94E-04
Choline chloride 139.62 7.02E+03 5.03E-02 D-Calcium pantothenate
238.27 8.79E+02 3.69E-03 Folic acid 441.4 2.08E+03 4.71E-03
i-Inositol 180.16 9.89E+03 5.49E-02 Niacinamide 122.12 1.59E+03
1.30E-02 Pyridoxine hydrochloride 205.64 1.57E+03 7.62E-03
Riboflavin 376.36 1.72E+02 4.56E-04 Thiamine hydrochloride 337.27
8.16E+03 2.42E-02 GROWTH FACTORS/PROTEINS GABA 103.12 1.01E+05
9.79E-01 Pipecolic Acid 129 1.27E+02 9.84E-04 bFGF 18000 1.04E+02
5.77E-06 TGF beta 1 25000 5.88E-01 2.35E-08 Human Insulin 5808
2.28E+04 3.92E-03 Human Holo-Transferrin 78500 1.08E+04 1.37E-04
Human Serum Albumin 67000 1.31E+07 1.95E-01 Glutathione (reduced)
307.32 1.96E+03 6.38E-03 OTHER COMPONENTS Hypoxanthine Na 136.11
1.61E+03 1.18E-02 Phenol red 354.38 5.99E+03 1.69E-02
Putrescine-2HCl 161.07 6.36E+01 3.95E-04 Thymidine 242.229 2.86E+02
1.18E-03 2-mercaptoethanol 78.13 7.66E+03 9.80E-02 Pluronic F-68
8400 1.96E+05 2.33E-02 Tween 80 1310 4.31E+02 3.29E-04
The systems containing a LN-521/e-cadherin substrate and mTeSR1
medium with additional albumin work extremely well for maintaining
the differentiated cells in their phenotype in a completely
chemically defined environment and xeno-free conditions without
feeders or any inhibitors of apoptosis.
It is contemplated that the cell culture medium will be completely
defined and xeno-free. The medium should also be devoid of any
differentiation inhibitors, feeder cells, or differentiation
inductors, or apoptosis inhibitors. Examples of feeder cells
include mouse fibroblasts or human foreskin fibroblasts. Examples
of differentiation inductors include Noggin or keratinocyte growth
factor.
The combination of the laminin substrate with the cell culture
medium of the present disclosure results in a cell culture system
that can be cheaper, yet provides higher efficiency in maintaining
differentiated cells. In essence, all that is required is a laminin
and a minimal amount of nutrition. It is particularly contemplated
that the laminin used in combination with this cell culture medium
is either LN-511 or LN-521.
The cell culture system in some embodiments includes at least one
of Laminin-411, Laminin-511, and Laminin-521 in the substrate, and
maintains differentiated human umbilical vein endothelial cells
(HUVECs) longer than shown by conventional fibronectin
substrates.
Primary Cell Transfection
The present disclosure also relates to methods for improving the
transfection efficiency with primary cells (i.e. differentiated
cells) and/or improving the survival rate of primary cells that
have been transfected.
As used herein, the term "vector" refers to a nucleic acid molecule
capable of transporting another nucleic acid to which it has been
linked. One type of vector is a "plasmid", which refers to a
circular double stranded DNA into which additional DNA segments may
be cloned. Another type of vector is a viral vector, wherein
additional DNA segments may be cloned into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors), are
integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host
genome. Moreover, certain vectors are capable of directing the
expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors"
or simply "expression vectors". In the present disclosure, the
expression of the laminin polypeptide sequence is directed by the
promoter sequences of the disclosure, by operatively linking the
promoter sequences of the disclosure to the gene to be expressed.
In general, expression vectors of utility in recombinant DNA
techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" may be used interchangeably,
as the plasmid is the most commonly used form of vector. However,
the disclosure is intended to include other forms of expression
vectors, such as viral vectors (e.g., replication defective
retroviruses, adenoviruses and adeno-associated viruses), which
serve equivalent functions.
The vector may also contain additional sequences, such as a
polylinker for subcloning of additional nucleic acid sequences, or
a polyadenylation signal to effect proper polyadenylation of the
transcript. The nature of the polyadenylation signal is not
believed to be crucial to the successful practice of the methods of
the disclosure, and any such sequence may be employed, including
but not limited to the SV40 and bovine growth hormone poly-A sites.
Also contemplated as an element of the vector is a termination
sequence, which can serve to enhance message levels and to minimize
readthrough from the construct into other sequences. Additionally,
expression vectors typically have selectable markers, often in the
form of antibiotic resistance genes, that permit selection of cells
that carry these vectors.
The primary cells can be transfected using any known transfection
method. Such methods include a baculovirus, a lentivirus,
lipofectamine, the calcium phosphate method, liposomes, cationic
polymers, electroporation, sonoporation, optical transfection, gene
electrotransfer, impalefection, hydrodynamic injection, gene gun,
magnetofection, and viral transduction. The vector is selected to
match with the transfection method.
The transfected primary cells are cultured upon the substrate that
contains the laminin. A cell culture system generally comprises a
substrate and a cell culture medium. The substrate provides a
support upon which the cells can grow. The medium provides the
nutrients to the cells. It is contemplated that the cell culture
medium will be completely defined and xeno-free. The medium should
also be devoid of any differentiation inhibitors, feeder cells, or
differentiation inductors, or apoptosis inhibitors. Examples of
feeder cells include mouse fibroblasts or human foreskin
fibroblasts. Examples of differentiation inductors include Noggin
or keratinocyte growth factor.
Normal transfection efficiency is about 10%. It is believed that a
higher efficiency rate can be obtained using the substrates
described herein.
The following examples are for purposes of further illustrating the
present disclosure. The examples are merely illustrative and are
not intended to limit devices made in accordance with the
disclosure to the materials, conditions, or process parameters set
forth therein.
EXAMPLES
A. HUVEC Cell Derivation
HUVECs were derived from umbilical cords according to a modified
protocol disclosed in Baudin et al., A protocol for isolation and
culture of human umbilical vein endothelial cells, Nat. Protoc.
2007; 2(3):481-5 (hereinafter "Baudin"). Collagenase A solution
from Roche in PBS buffer was briefly injected into pre-washed vein
of human umbilical cord. Collagenase was incubated at 37 degrees
Celsius, and then washed away with cell suspension. The cell
suspension was plated on laminin-coated plates.
AI. Laminin Coating
Primary HUVECs were cultured on top of different substrate coatings
including conventional fibronectin as a control and recombinant
laminins, specifically human recombinant LN-411, LN-511, LN-521,
LN-111, and LN-211, either alone or in combination. Combinations of
LN-411/LN-511, LN-511 alone, and LN-521 alone showed successful
long-term culture of HUVEC cells in vitro.
Laminin substrate coatings were stored in PBS at -70 degrees
Celsius, were thawed on wet ice (approximately 4 degrees Celsius)
and then dissolved in sterile PBS to a concentration of 5
micrograms per milliliter (ug/ml). 80 microliters (uL) of substrate
solution was used to coat 96-well plates overnight at 4 degrees
Celsius or for 2 hours at 37 degrees Celsius in a cell culture
incubator. The wells of the 96-well plates were pre-washed with PBS
buffer prior to plating the cells.
AII. Culturing
HUVECs were cultured in sterile incubators, each with the
temperature set to 37 degrees Celsius and CO.sub.2 levels set to
5%. Sarstedt 96-cell plates were used for culturing, with an added
medium amount of 200 uL/well. The medium composition used, as
disclosed in Baudin, was filtered through a 0.22 micrometer (um)
filter and stored at 4 degrees Celsius for about 2-3 weeks.
AIII. Cell Passaging
HUVEC cells were passaged in vitro at several densities. HUVEC
cells were split into 1:5, 1:10, or 1:20 splits using standard
Trypsin-EDTA solution from Gibco. Trypsin-EDTA was applied
pre-warmed for about 3-5 minutes. Trypsin was inhibited by serum
within the cell culture medium.
AIV. HUVEC Cell Analysis
HUVECs were first characterized by quantification methods. This
included recorded and quantifying HUVECs using the Operetta machine
from Perkin-Elmer. Magnification was chosen at 10.times.,
20.times., and 40.times..
Immunocytochemistry analysis was subsequently performed on HUVECs
by pre-washing adherent cells twice with warm PBS buffer, applying
100 uL of 4% paraformaldehyde (PFA) solution, incubating at room
temperature for 20 minutes, then washing the wells three times with
PBS. Fixed cells were permeabilized by 0.1% Triton-X solution at
room temperature for 15 minutes, then washed by PBS buffer three
times and blocked by 10% bovine fetal serum in PBS, supplemented by
0.1% Tween stored for 30 minutes at room temperature or overnight
at 4 Celsius.
HUVECs typically express the endothelial marker known as von
Willebrand factor (vWF) after 4 passages. Therefore, vWF factor was
used to define positive cells belonging to the endothelial cell
type. Smooth Muscle Actin (SMA) was used as a negative marker to
define fibroblasts or fibroblast-like differentiated cells within
in vitro population. DAPI was used to stain the nuclei, which is
necessary to define Total Cell Population using cell population
analysis on the Operetta machine using Harmony software provided by
Perkin-Elmer. Rhodamine-phalloidine conjugates were used to
visualize f-actin, which acts as a marker of cytoskeleton structure
and cell borders.
Quantitative RT-PCR was performed to compare quantitative levels of
positive marker (vWF factor) versus negative marker (SMA) in HUVEC
cultures in vitro after long passaging.
FIG. 3 is photomicrograph of human umbilical vein endothelial cells
(HUVECs) grown on a fibronectin (FNE) substrate, 10.times.
magnification, after 5 passages, with expression of von Willebrand
factor (vWF), f-actin, and DAPI. FIG. 4 is a photomicrograph of
HUVECs grown on a LN-521 substrate, 10.times. magnification, after
5 passages, with expression of vWF, f-actin, and DAPI. FIG. 5 is a
photomicrograph of HUVECs grown on a LN-521 substrate, 10.times.
magnification, after 7 passages, with expression of vWF, f-actin,
and DAPI. In these three figures, the von Willebrands factor (vWF)
is stained and appears as a green color. As seen here, the HUVECs
grown on fibronectin substrate (FIG. 3) do not express the
endothelial marker as well as cells on grown on LN-521 substrate
(FIG. 4 and FIG. 5), as seen by more green color expression of vWF
factor. FIG. 3 has much more black, yellow, and blue color compared
to FIG. 4 and FIG. 5, which are almost completely green. These
figures show that LN-521 effectively prevents dedifferentiation
better than conventional fibronectin substrate. The endothelial
cells grow equally well on the fibronectin substrate, but do not
maintain their phenotype as well.
FIG. 6 is a photomicrograph of HUVECs grown on a LN-411/511
substrate, 10.times. magnification, after 5 passages, with
expression of vWF, f-actin, and DAPI. FIG. 7 is a photomicrograph
of HUVECs grown on a LN-511 substrate, 10.times. magnification,
after 5 passages, with expression of vWF, f-actin, and DAPI. The
vWF factor shows greater expression in cells grown on LN-511
relative to cells grown on LN-411/511 substrate. In FIG. 6, the
majority of the picture is black or yellow. In FIG. 7, there are
many more cells and each cell has some green color. However, both
FIG. 6 and FIG. 7 show less vWF expression than seen for cells on
LN-521 substrate in FIG. 4 and FIG. 5.
FIG. 8 is graph of RNA Gene Expression showing Acta2 gene
expression (negative marker) and vWF gene expression (positive
marker). The data was obtained by performing qRT-PCR according to
standard procedures. The Acta2 data is on the left side. The five
columns are labeled, going from left to right, according to the
table below. Note that Ln 08 refers to laminin-411, Ln 10 refers to
laminin-511, and Ln 11 refers to laminin-521. Ln 08_10 is a mixture
of laminin-411/511.
TABLE-US-00006 Column Text Value Left red Fibronectin 1.000 Left
green Ln 08_10 0.236 Left blue Ln 10 0.255 Right red Ln 11 1.640
Right green Ln 11_less_cells 1.674
The vWF data is on the right side of FIG. 8. The five columns are
labeled, going from left to right, according to the table
below.
TABLE-US-00007 Column Text Value Left red Fibronectin 1.000 Left
green Ln 08_10 0.570 Left blue Ln 10 1.364 Right red Ln 11 0.811
Right green Ln 11_less_cells 0.994
FIG. 9 is a graph of quantified percentage of vWF-positive HUVECs
within a population after a long-term culture of HUVECs on human
recombinant laminin-521. FIG. 10 is a graph of quantified
percentage of vWF-positive HUVECs within a population after a
long-term culture of HUVECs on human recombinant Fibronectin. With
reference to FIG. 9 and FIG. 10, the percentage of vWF-positive
HUVECs is stable after 7 passages (approximately 20.3 doublings)
when Laminin-521 is used as the substrate. By contrast, the
percentage of vWF-positive HUVECs is not stable at 5 and 7 passages
when traditional fibronectin is used as the substrate. Therefore,
recombinant LN-521 substrate enables HUVECs to retain their
phenotype longer than when plated on fibronectin substrate.
FIG. 11 is a proliferation curve showing the proliferation of
HUVECs on different substrate coatings, dependent on days in
culture up through 160 days. LN-521 always had the highest number
of doublings. LN-511 and LN-411/511 also supported a high number of
doublings up to around 65 days in culture. Around that time, their
advantage over fibronectin (FNE) began to shrink.
B. Mouse Pancreatic Insulin-Producing Islet Beta Cell
Derivation
Murine islet cells were derived according to the modified protocol
by Dong-Sheng Li et. al., as published in "A protocol for islet
isolation from mouse pancreas," Nature Biotechnology 2009. All
manipulation with mouse pancreas was performed under a dissection
microscope, with a corresponding magnification of 0.63. All
instruments in contact with mouse pancreas were sterilized with
ethanol solution. The bile pathway to the duodenum in mouse
subjects was blocked by clamping ampula with surgical clamps.
A 30 gauge, one-half inch needle, was inserted into the joint site
of hepatic duct and cystic duct and inserted until reaching the
middle of common bile duct. Collagenase A (available from Roche)
was used at a concentration of 5 mg/ml. Collagenase A was slowly
injected into murine pancreas up to volume of 3 ml and thereby
inflating the pancreas. Inflated pancreas was removed and soaked in
2 ml of collagenase A solution. Pancreas in collagenase A solution
was transferred to sterile 50 ml Falcon tube, incubated at
temperature of 37.degree. C. in water bath, and shaken every 5
minutes for better spreading of collagenase. After 18-25 minutes of
incubation in water bath, the pancreas was substantially
dissociated.
Digestion was terminated by putting the tube on ice and adding 25
ml of ice cold buffer. In order to remove exocrine cells and
collagenase solution, the islet cells were repeatedly washed and
centrifuged. The resulting suspension of cells was centrifuged at
290 g for 30 seconds at 4.degree. C. and supernatant was discarded.
The remaining pellet was resuspended with 20 ml ice-cold buffer,
centrifuged again at 290 g for 30 seconds at 4.degree. C., and the
supernatant was discarded. The resulting pellet was resuspended
with 15 ml of buffer and poured onto a prewetted 70 micrometer
(.mu.m) cell strainer. The tube was washed with 20 ml of buffer and
poured again onto the strainer. Islet cells from the strainer were
rinsed with islet culture medium into a 100-mm tissue culture Petri
dish. Lastly, the islet cells were hand-picked and transferred to
another 100-mm Petri dish.
BI. Islet Beta Cell Depletion
In order to completely remove the remains of exocrine tissue and
non-islet connective tissue from islet culture, the islet beta
cells were cultured for 2-3 days in 100-mm Petri dish, which
allowed the cells and cell aggregates to settle. After that the
islets were evaluated for having a smooth, round shape that was
free from debris. Selected islets were hand-picked and plated onto
another 100-mm Petri dish.
BII. Transferring Mouse Islets onto Recombinant Human
Laminin-Coated Tissue Culture-Grade Plates
Tissue-culture grade 96-well plates, e.g. available from
Perkin-Elmer Cell Carrier plates or Sarstedt, were coated with
solutions of human recombinant laminins suspended in PBS solution
for over 2 hours at 37 C (stored in cell incubator) or for over 20
hours at 4 C. 96-well plates were washed 2-3 times with PBS buffer
before use. Islet culture medium was input into the wells prior to
islet plating, and was then equilibrated in an incubator. Islet
cells were hand-picked and plated onto laminin-coated plates.
Culture medium was changed every 2-3 days.
BIII. Analysis of Islet Beta Cells
Islet cells were analyzed according to three different
methodologies. FIGS. 12-18 pertain to analysis of beta cell
morphology, which includes the use of a phase-contrast microscope
at magnification of .times.4, .times.10, .times.20 and .times.40.
FIG. 19 pertains to analysis of beta cell immunohistochemistry,
specifically involving an antibody against C-peptide, a marker of
insulin expression. FIG. 20-21 pertains to analysis of cell
proliferation by EdU staining. The EdU molecule incorporates into
DNA strands of nuclei of cells that have divided.
Rhodamine-phalloidine, used for cytoskeleton structure analysis,
and Hoechst, used to stain the nuclei of cells, were also used in
combination with anti-C-peptide and/or EdU for additional
information.
Mouse islets were cultured on human recombinant laminins in order
to imitate the natural "environmental niche" for beta cells and to
enable the beta cells to grow as a syncytium. When .alpha.5 chain
laminins were used as coatings in vitro, beta cells were able to
express insulin genes. The result was that different types of
laminins exerted different effects on mouse pancreatic islets, e.g.
the ability to produce insulin or proliferate.
Laminins 411, 511, 111, and 521 were coated on a surface before
depositing islets. FIGS. 12-16 show the results. FIG. 12 shows the
islets on a surface coated with LN-521. FIG. 13 shows the islets on
an uncoated surface. FIG. 14 shows the islets on a surface coated
with LN-411. FIG. 15 shows the islets on a surface coated with
LN-511. FIG. 16 shows the islets on a surface coated with LN-111.
Desirably, the islet would adhere and spread to the surface.
As seen in FIG. 12, LN-521 provided a robust, long-lasting effect
of islet adhering uniformly and spreading upon the culture plate
surface. The islet of FIG. 13 did not adhere well on an uncoated
surface, as seen in its generally circular shape. With reference to
FIG. 14, the islet plated on LN-411 did not have a uniform shape of
adhesion. The right side adhered, but the left side failed to
adhere. With reference to FIG. 15, the islets plated on LN-511 had
different behavior, or in other words were inconsistent. One islet
adhered, while one did not. Looking at FIG. 16, the islets plated
on LN-111 failed to adhere and spread. The effect of LN-521 on
islets may be due to a specific molecule interaction or
characteristic.
With reference to FIGS. 17-18, a specific effect on the morphology
of the islets is demonstrated by comparing islets deposited upon
laminin-521 in FIG. 17 to islets deposited upon laminin-111 in FIG.
18. Mouse pancreatic insulin-producing beta islets expanded on
human recombinant laminin-521, whereas islets expanded on
laminin-111 retained their three-dimensional spherical shape.
Differences in morphology and in the cell community infrastructure
can be seen on either low magnification (.times.10) (FIGS. 17(a)
and 18(a)) or high magnification (.times.40) (FIGS. 17(b) and
18(b)).
With reference to FIG. 19, after 3-4 weeks in culture, beta islets
were deposited upon laminin-521 and stained positively for
C-peptide, which is a marker for insulin expression. FIG. 19(a)
shows a positive indication of C-peptide, and therefore successful
production of insulin, when beta islets are deposited on
laminin-521. FIG. 19(b) shows the positive indication of C-peptide,
as well as the nucleus of beta islets on laminin-521, shown through
blue colored Hoechst indicator (the upper right dot).
With reference to FIG. 20, after 3-4 weeks in culture, beta islets
deposited on laminin-521 maintained capacity for proliferation.
With reference to FIG. 20(a), beta islets demonstrated positive EdU
staining, shown by a green color (the solid dots) in the nuclei of
proliferated cells. The nuclei are spread out relative to the beta
islets in FIG. 19. In FIG. 20(b), the green color EdU stain was
merged with a phase-contrast photograph for additional context on
the location of proliferated nuclei.
In FIG. 21, the joint effect of proliferation of islets cultured
upon laminin-521 and expression of C-peptide within the same islets
is demonstrated. With reference to FIG. 21(a), mouse pancreatic
insulin-producing beta islet cells when cultured on human
recombinant laminin-521 maintained proliferation potential and
expressed C-peptide. This was demonstrated by the contemporaneous
positive green EdU staining as well as the orange color (indication
of c-peptide, marker of insulation production) in FIG. 21(a). The
green EdU stains were the sharp bright points, while the orange
color was the portions surrounding the sharp points. In FIG. 21(b),
blue Hoechst dye was localized in the nuclei of all islets, while
green EdU indicated the nuclei of islets divided during the last
three days. The islets in FIG. 21(b) that have divided during the
last three days are farther spaced apart than the nuclei of all
islet cells shown by Hoechst. The blue nuclei were the extremely
bright spots, while the green nuclei were the less bright spots
around the perimeter.
The present disclosure has been described with reference to
exemplary embodiments. Obviously, modifications and alterations
will occur to others upon reading and understanding the preceding
detailed description. It is intended that the present disclosure be
construed as including all such modifications and alterations
insofar that they come within the scope of the appended claims or
the equivalents thereof.
* * * * *